International Journal of ChemTech Research
CODEN (USA): IJCRGG
ISSN: 0974-4290
Vol.8, No.4, pp 1810-1816,
2015
Characterization and Antioxidant Activity ofNon-Polar
Extractfrom Crude Palm Oil and Palm Methyl Ester
Ahmad Gazali Sofwan Sinaga1* and Donald Siahaan1
1
Indonesian Oil Palm Research Institue, Jl. BrigjenKatamso No. 51 Medan,
North Sumatera, Indonesia
Abstract: Palm oil has many minor components that can act as natural antioxidant. It
containing carotenoid and vitamin E. This research was conducted to determine antioxidant
activity ofnon polar extract from crude palm oil and fatty acid methyl ester. The oil extract
obtained from crude palm oil by solvent extraction with hexane (CPO) and transesterification
method followed solvent extraction with hexane (PME). Carotene content from non-polar
extracts were analyzed by using UV-visible spectrophotometer, while carotene composition
(α- and β-carotene) and vitamin E (tocopherol and tocotrienol) compositions were analyzed
by using high performance liquid chromatography. Glycerides and esters content was
analyzed by gas chromatography. Antioxidant activity of oil extract was determined by using
2,2’-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay method. Result
revealed that PME has higher content carotenoid and vitamin E than CPO. As expected, the
concentration of carotenoid and vitamin E in PME increased with transesterification process.
Results also showed that all of non-polar extracts exhibited antioxidant activity significantly,
as proven by inhibitory concentration 50% (IC50) of PME and CPO is 5.9 µg/ml and 15.6
µg/ml. It is suggested that the presence of carotenoid and vitamin E may have a potential
effect as natural antioxidant.
Keywords: Crude palm oil, palm methyl ester, carotenoid, vitamin E, antioxidant.
Introduction
Crude palm oil is a vegetable oil containing minor components such as carotenoids (500-700 µg/ml)
and vitamin E (600-1000 µg/ml).1 CPO has a significant amount of carotene that can be isolated by various
methods. Some researchs has developed carotene isolation process from palm oil such as solvent extraction,
transesterification,2 saponification,3 adsorption, membrane,4 and solvolytic micellization.4 Transesterification is
general term used to describe transformed ester form into other through interchange of the alkoxy group. This
reaction will produce esters (PME), glycerol and carotene.6
Carotene is tetraterpene that characterized by a conjugated system of double bonds. The extreme
hydrophobic character of carotenes has function as antioxidant and play important roles in protection of body
tissues from damage cause by free radicals.7 Carotene and vitamin E has potential as a supplement, as well as a
source of antioxidants in pharmaceutical preparations such as creams, ointments and gels. 8 Yeh and Hu,9
mentioned carotene effective as lung cancer drug, degenerative eye disease and cataract,10 and decrease blood
glucose level.11
Antioxidants have used to preserve fats and oils without degradation. These substances inhibit oxidative
damage in oil content.12 Several methods had developed to measure the free radical scavenging activity.13
DPPH (2,2’-diphenyl-1-picrylhydrazyl) free radical scavenging assay method is common applied in
Ahmad Gazali Sofwan Sinaga et al /Int.J. ChemTech Res. 2015,8(4),pp 1810-1816.
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determining antioxidant activity of natural product. This assay method also used to study the scavenging
activity of antioxidant in oils. Until now, IOPRI not yet determine the ability of free radical reduction activity of
carotene from CPO and PME. These researches describe about free radical scavenging activity of carotene
extract from CPO and PME.
Experimental
Reagents and Standards
All the solvents used for sample preparation and extraction were of analytical grade obtained from
Merck (Darmstadt, Germany). All solvents used for HPLC analysis and UV-visible analysis were obtained from
Merck (Darmstadt, Germany). β-carotene standards, tocopherol and tocotrienol standards were purchased from
Sigma Chemical Co. (Sigma-Aldrich Company, St. Louis, MO, USA).
Instrumentation
Total of carotenoid contents was analysed by using spectrophotometer UV-visible (1700, Shimadzu). αand β-carotene composition and vitamin E (tocopherol and tocotrienol) analysed by using HPLC (Perkin
Elmer), equipped with a YMC (Tokyo, Japan) C30 column (250 x 4,6 mm I.D., 5 μm particle size) and Agilent
Technologies (Santa clara, USA) C18 Column (4,6 x 150 mm, 2,7 μm particle size), respectively. Glyceride and
ester contents were analyzed by using gas chromatography (GC-14B, Shimadzu), equipped with DB-5 HT
capillary column (0,53 mm x 5 m).
Extraction Process of Carotenoids
Extraction from CPO
The carotene extraction process of CPO using solvent extraction according to Ahmad et al, 14 with
slightly modification. CPO and hexane were mixed at a ratio of 1:5 (CPO:hexane) in vortex for 10 minutes. The
product was extracted carotene from CPO and used in the next analysis.
Extraction from PME
Production of PME by using transesterification process according to Panjaitan et al,3 CPO and methanol
were mixed at a ratio 1:9 (CPO:methanol) with KOH as a base catalyst for 60 minutes at 60°C. The glycerol
produced was removed while the methyl ester obtained washed with water and hexane. The rich carotene in
hexane layer collected and used in the next analysis.
Analysis of Non-Polar Extract
HPLC Analysis of Carotenoids
Identification of α- and β-carotene composition from CPO and PME analysed by using HPLC
according to Strati et al,15 with slightly modification.
HPLC Analaysis of Total Vitamin E (Tocopherol and Tocotrienol)
Identification of total tocopherol and tocotrienol from CPO and PME were analyzed by using HPLC
according to Ahmadi et al,16with slightly modification.
Spectrophotometer UV-visible Analysis of Total Carotenoids
Extracted carotene from CPO and PME were analyzed by using spectrophotometer UV-visible at 446
nm according to MPOB test method P2.6: 2004.17
Gas Chromatography of Glyceride and Ester Contents
Analysis of glycerides and ester content were prepared according to MPOB test method C2.11 by using
gas chromatography.17
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Analysis of DPPH Radical Scavenging Activity
Antioxidant activity of carotenes from CPO and PME were analyzed by scavenging activity of stable
DPPH. This method was according to Rubalya and Neelamegan,13 with slightly modification. The carotene was
examined with six different concentration (1.5 – 9.0 µg/ml) with hexane and chloroform as solvent at ratio 2:3.
Amount 0,5 ml mixed solution from each concentration were placed into tube with adding 4 ml 0,5 mM of
DPPH ethanolic solution. The mixture measured by using spectrophotometer UV-visible at 515 nm.
Antioxidant activity calculated by plotting percentage inhibition against different concentrations. Inhibitor
concentration (IC50) is an antioxidant concentration that inhibits the DPPH reaction by 50% under experimental
conditions.
Results and Discussion
HPLC Analysis of Carotenoids in CPO and PME
Figure 1. Carotenes Composition from CPO
Figure 2. Carotenes Composition from PME
The HPLC chromatogram of α- and β-carotene composition using HPLC presented in Figure 1 and
Figure 2. Based on those results, both contain high β-carotene about 66.6% and 64.6% for CPO and PME
respectively, whereas α-carotene about 34.3% and 35.4% for CPO and PME respectively. It shows that the CPO
and PME contain the high β-carotene. These results are consistent with the study by Panpipat and Chaijan,18
who have reported that β-carotene and α-carotene is the major component contained in palm about 80-90% of
the total carotenoids. According to Sthal and Sies,19 β-carotene is a pro-vitamin A, which can be utilized as a
source of vitamin A. Thus, high levels of β-carotene on CPO and PME chance as the largest producer of
vitamin A.
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Spectrofotometer UV-visible Analysis of Total Carotenes
Table 1. Characteristic of carotene extract from CPO and PME
Parameters
Triglyceride (%)
Diglyceride (%)
Monoglyceride (%)
Ester (%)
Carotene (µg/ml)
nd: not detected
Crude Palm Oil
76.84
14.37
nd
11.41
510
Palm Methyl Ester
nd
nd
0,17
93.80
599
The results of the carotene concentration analysis using UV-visible spectrophotometer presented in
Table 1. The result shows that carotene extract from PME achieved 599 µg/ml, it is higher than CPO which
achieved only 510 µg/ml. Carotene in CPO still highly bound to the triglyceride so that carotene extract lower
than PME that do not contain triglyceride. It was proven in PME only containing monoglycerides, diglycerides
and triglycerides under 0,5%. Based on the study by Hasibuan et al,20 the transesterification method has been
successfully transformed glyceride into ester so that carotene more soluble in the solvent.
Gas Chromatography Analysis of Glycerides and Esters
Figure 3. ChromatogramGlycerides and Esters Content of CPO
Ahmad Gazali Sofwan Sinaga et al /Int.J. ChemTech Res. 2015,8(4),pp 1810-1816.
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Figure 4. ChromatogramGlycerides and Esters Content of PME
The results of the glycerides and esters content analysis of CPO and PME presented in Figure 3 and
Figure 4. Results shows that CPO contains high levels of triglycerides about 76.84%, whereas PME does not
contain triglycerides. The high triglyceride concentration can affect the purity of carotene extracted according to
Panjaitan et al,3 triglyceride very strong binds to carotene, so thatmake difficult to obtain pure carotene with
high triglyceride concentrations. Meanwhile, the PME does not contain triglycerides but contain high levels of
esters about 93.80%. This happens because the transesterification process has turned into a glyceride ester
compound.6
HPLC Analaysis of Total Vitamin E (Tocopherol and Tocotrienol)
Table 2. Total vitamin E (Tocopherol and Tocotrienol) Composition
Extract Sources
Crude Palm Oil
Palm Methyl Ester
nd: not detected
Tocopherol (%)
nd
0.12
Tocotrienol (%)
14.16
83.26
The results of total vitamin E (tocopherol and tocotrienol) contents from CPO and PME presented in
Table 2. Results shows that CPO has no content tocopherol and tocotrienol about 14.16%, whereas PME has
tocopherol and tocotrienol about 0.12% and 83.26%, respectively. Extract from CPO obtained little amount of
tocotrienol, while tocopherol was not detected. This can occur due to the involvement of the high triglyceride
contents, possibly that can interfere extraction process using non polar solvent. Extract from PME contains high
tocopherol and tocotrienol, which shown in the process of more than 90% triglycerides has transformed into
ester.20
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DPPH Radical Scavenging Activity
Figure 5. DPPH radical-scavenging activity of carotene extract from CPO and PME
Table 3. DPPH antioxidant activity of carotene extract from CPO and PME
Concentrations
(µg/ml)
1.5
3
4.5
6
7.5
9
Antioxidant Activity (%)
CPO
PME
7.5
15.9
12.3
26.4
17.9
36.5
22.6
54.8
24.8
63.9
30.1
69.3
Table 3 shows free radical activity of carotene extract from CPO and PME. The results indicate that
antioxidant activity of carotene extract from PME stronger than CPO. Figure 5 shows that antioxidant activity
of crude palm oil at 9 µg/ml was 30.1% lower than PME was 69.3%. The IC 50 results showed PME stronger
than crude palm oil approximately 5.9 µg/ml and 15.6µg/ml, respectively. According Sinaga et al, 20 high IC50
values was below 50 µg/ml. Some studies reported that high levels of α-carotene and β-Carotene could increase
antioxidant activity of a product.18 Based on the HPLC results, β-carotene concentrations from palm oil was
higher than PME. However, the antioxidant activity of PME stronger than crude palm oil. This expected, due to
changes trans-β-carotene to cis-β-carotene isomers during transesterification process. Levin and Mokady,22
reported that 9-cis-β-carotene has a higher antioxidant potency than that of the all-trans isomer.
Discussion
Carotene concentrate from PME has antioxidant activity stronger than CPO. Possibility carotene does
not act as free radical scavenger directly due to high triglyceride content. However, both sources of carotene
concentrate has high antioxidant activity and further we conclude that carotene from palm oil is a potential
candidate for natural antioxidant.
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